Effects of heat and pesticide stress on life history, physiology and the gut microbiome of two congeneric damselflies that differ in stressor tolerance.

The combined impact of toxicants and warming on organisms is getting increased attention in ecotoxicology, but is still hard to predict, especially with regard to heat waves. Recent studies suggested that the gut microbiome may provide mechanistic insights into the single and combined stressor effects on their host. We therefore investigated effects of sequential exposure to a heat spike and a pesticide on both the phenotype (life history and physiology) and the gut microbiome composition of damselfly larvae. We compared the fast-paced Ischnura pumilio, which is more tolerant to both stressors, with the slow-paced I. elegans, to obtain mechanistic insights into species-specific stressor effects. The two species differed in gut microbiome composition, potentially contributing to their pace-of-life differences. Intriguingly, there was a general resemblance between the stressor response patterns in the phenotype and in the gut microbiome, whereby both species responded broadly similar to the single and combined stressors. The heat spike negatively affected the life history of both species (increased mortality, reduced growth rate), which could be explained not only by shared negative effects on physiology (inhibition of acetylcholinesterase, increase of malondialdehyde), but also by shared effects on gut bacterial species' abundances. The pesticide only had negative effects (reduced growth rate, reduced net energy budget) in I. elegans. The pesticide generated shifts in the bacterial community composition (e.g. increased abundance of Sphaerotilus and Enterobacteriaceae in the gut microbiome of I. pumilio), which potentially contributed to the relatively higher pesticide tolerance of I. pumilio. Moreover, in line with the response patterns in the host phenotype, the effects of the heat spike and the pesticide on the gut microbiome were mainly additive. By contrasting two species differing in stress tolerance, our results suggest that response patterns in the gut microbiome may improve our mechanistic understanding of single and combined stressor effects.

Synergistic survival-related effects of larval exposure to an aquatic pollutant and food stress get stronger during and especially after metamorphosis and shape fitness of terrestrial adults.

To improve the ecological risk assessment of aquatic pollutants it is needed to study their effects not only in the aquatic larval stage, but also in the terrestrial adult stage of the many animals with a complex life cycle. This remains understudied, especially with regard to interactive effects between aquatic pollutants and natural abiotic stressors. We studied effects of exposure to the pesticide DNP (2,4-Dinitrophenol) and how these were modulated by limited food availability in the aquatic larvae, and the possible delayed effects in the terrestrial adults of the damselfly Lestes viridis. Our results revealed that DNP and low food each had large negative effects on the life history, behaviour and to a lesser extent on the physiology of not only the larvae, but also the adults. Food limitation magnified the negative effects of DNP as seen by a strong decline in larval survival, metamorphosis success and adult lifespan. Notably, the synergism between the aquatic pollutant and food limitation for survival-related traits was stronger in the non-exposed adults than in the exposed larvae, likely because metamorphosis is stressful itself. Our results highlight that identifying effects of aquatic pollutants and synergisms with natural abiotic stressors, not only in the aquatic larval but also in the terrestrial adult stage, is crucial to fully assess the ecological impact of aquatic pollutants and to reveal the impact on the receiving terrestrial ecosystem through a changed aquatic-terrestrial subsidy.

Thermal and latitudinal patterns in pace-of-life traits are partly mediated by the gut microbiome.

The integration of life-history, physiological and behavioural traits into the pace-of-life generates a powerful framework to understand trait variation in nature both along environmental gradients and in response to environmental stressors. While the gut microbiome has been hypothesized as a candidate mechanism to underlie differentiation in the pace-of-life, this has been rarely studied. We investigated the role of the gut microbiome in contributing to the differentiation in pace-of-life and in thermal adaptation between populations of Ischnura elegans damselfly larvae inhabiting warmer low latitudes and colder high latitudes. We carried out a common-garden experiment, whereby we manipulated the exposure of the damselfly larvae to two key global warming factors: 4 °C warming and a 30 °C heat wave. Comparing the bacterial composition of the food source and the bacterioplankton indicated that damselfly larvae differentially take up bacteria from the surrounding environment and have a resident and functionally relevant microbiome. The gut microbiome differed between larvae of both latitudes, and this was associated with the host's latitudinal differentiation in activity, a key pace-of-life trait. Under heat wave exposure, the gut microbial community composition of high-latitude larvae converged towards that of the low-latitude larvae, with an increase in bacteria that likely are important in providing energy to cope with the heat wave. This suggests an adaptive latitude-specific shift in the gut microbiota matching the better ability of low-latitude hosts to deal with heat extremes. In general, our study provides evidence for the gut microbiome contributing to latitudinal differentiation in both the pace-of-life and in heat adaptation in natural populations.

Evolution of pesticide tolerance and associated changes in the microbiome in the water flea Daphnia magna.

Exposure to pesticides can have detrimental effects on aquatic communities of non-target species. Populations can evolve tolerance to pesticides which may rescue them from extinction. However, the evolution of tolerance does not always occur and insights in the underlying mechanisms are scarce. One understudied mechanism to obtain pesticide tolerance in hosts are shifts toward pesticide-degrading bacteria in their microbiome. We carried out experimental evolution trials where replicated experimental populations of the water flea Daphnia magna were exposed to the pesticide chlorpyrifos or a solvent control, after which we performed acute toxicity assays to evaluate the evolution of chlorpyrifos tolerance. Additionally, we quantified changes in the microbiota community composition of whole body and gut samples to assess which sample type best reflected the pesticide tolerance of the Daphnia host. As expected, chlorpyrifos-selected clones became more tolerant to chlorpyrifos as shown by the higher EC5048 h (36% higher) compared with the control clones. This was associated with shifts in the microbiome composition whereby the abundance of known organophosphate-degrading bacterial genera increased on average ~4 times in the chlorpyrifos-selected clones. Moreover, the abundances of several genera, including the organophosphate-degrading bacteria Pseudomonas, Flavobacterium and Bacillus, were positively correlated with the EC5048 h of the host populations. These shifts in bacterial genera were similar in magnitude in whole body and gut samples, yet the total abundance of organophosphate-degrading bacteria was ~6 times higher in the whole body samples, suggesting that the gut is not the only body part where pesticide degradation by the microbiome occurs. Our results indicate that the microbiome is an important mediator of the development of tolerance to pesticides in Daphnia.

Evolution of tolerance to chlorpyrifos causes cross-tolerance to another organophosphate and a carbamate, but reduces tolerance to a neonicotinoid and a pharmaceutical.

Exposure to pesticides is a major stressor in freshwater ecosystems. While populations can evolve tolerance to pesticides and thereby ensure their persistence in contaminated environments, this may have important consequences for their sensitivity to other pollutants. Indeed, tolerance to one pollutant may both increase (as a cost of tolerance) or decrease (cross-tolerance) the sensitivity to other pollutants. Despite the increasing concern of pharmaceuticals in waterbodies, no patterns of pesticide-induced (cross-)tolerance have been studied. We conducted 48 h acute toxicity assays with a range of concentrations of different pollutants to determine how the evolution of tolerance to the insecticide chlorpyrifos affects the sensitivity to other pesticides and a pharmaceutical in the water flea Daphnia magna, a keystone zooplankton species in aquatic food webs. We capitalized on an experimental evolution trial with chlorpyrifos, hence could unambiguously identify any patterns in increased tolerance or sensitivity to the other pollutants as a direct result of the evolution of tolerance to chlorpyrifos. We found that evolution of tolerance to chlorpyrifos conferred cross-tolerance to another organophosphate, namely malathion (mean change in EC50,48h: factor 3.1), and to the carbamate carbaryl (factor 1.7), confirming that a shared mode of action favours the evolution of cross-tolerance. While the evolution of tolerance to chlorpyrifos did not affect the sensitivity to the pyrethroid esfenvalerate, it increased the sensitivity to the neonicotinoid imidacloprid as shown by the decrease in EC50,48h (factor 0.6). Notably, we demonstrated for the first time that the evolution of tolerance to a pesticide increased the sensitivity to a pharmaceutical, namely fluoxetine (decrease in EC50,48h with factor 0.7), thereby identifying an overlooked cost of tolerance to a pesticide. Given the increasing exposure to pesticides and pharmaceuticals, our results highlight that considering cross-tolerance and costs of tolerance is crucial in risk assessment of both pesticides and pharmaceuticals in aquatic ecosystems.

The pace-of life explains whether gills improve or exacerbate pesticide sensitivity in a damselfly larva.

Trait-based approaches are promising to make generalizations about the sensitivity of species and populations to pesticides. Two traits that may shape the sensitivity to pesticides are the surface area (related to pesticide uptake) and the metabolic rate (related to pesticide elimination). We compared the sensitivity of damselfly larvae to the pesticide chlorpyrifos and how this was modified by loss of external gills (autotomy, reducing the surface area) in both fast pace-of-life (high metabolic rate) and slow pace-of-life (low metabolic rate) populations of Ischnura elegans. The slow-paced populations were more sensitive to the pesticide than the fast-paced populations in terms of survival, growth and energy metabolism. This suggests the higher metabolic rate of fast-paced populations enabled a faster pesticide elimination. Pesticide exposure also reduced heat tolerance, especially in slow-paced larvae under hypoxia. Gill loss had opposite effects on pesticide sensitivity in slow- and fast-paced populations. In slow-paced larvae, gill loss lowered the sensitivity to the pesticide, while in fast-paced larvae, gill loss increased the sensitivity. This difference likely reflects the balance between the roles of the gills in pesticide uptake (more detrimental in slow-paced populations) and oxygen uptake (more important in fast-paced populations). Our results highlight the need to consider trait interactions when applying trait-based approaches to predict the sensitivity to pesticides.

The impact of salinity on a saline water insect: Contrasting survival and energy budget.

Water salinity is a major driver of aquatic insects' distribution. Saline species are usually generalists with high survival and performance at both low and high salinity levels. Yet, costs of high salinity may be underestimated as these are most often measured in terms of larval life history traits, while effects of larval stressors may only be detectable when looking at physiological traits and traits in the adult stage. Here, we assessed the lethal and sublethal physiological effects of embryonic and larval exposure to a range of salinity levels in the damselfly Lestes macrostigma, both during and after metamorphosis. This species inhabits temporary freshwaters where salinity increases during the drying phase. Salinity had no effect on egg hatching success within the range 2-9.5 g/L sea salt (conductivity range 3.45-14.52 mS/cm). With increasing salinity (up to 16 g/L, 23.35 mS/cm), growth rate decreased and larvae took longer to emerge and did so at a smaller size. Larval survival to metamorphosis increased with salinity up to 8 g/L (12.45 mS/cm) and then declined at 16 g/L. Exposure to salinity in the larval stage had no effect across metamorphosis on both the adult thorax muscle mass and flight performance, and the investment in immune function. Increasing salinity in the larval stage also had no effect on the energy available but increased the energy consumption in the adult stage, resulting in a lower net energy budget. These negative sublethal effects of increasing salinity hence bridged metamorphosis and contrasted with the mortality data, suggesting that the higher mortality at the low salinity levels selected for larvae with the best body condition. Our results highlight the importance of taking into account other life-history and physiological traits, besides mortality, ideally across different life stages, to better understand and predict consequences of increasing salinization on freshwater insects.

Daily temperature variation lowers the lethal and sublethal impact of a pesticide pulse due to a higher degradation rate.

Daily temperature variation (DTV) is an important warming-related stressor that may magnify pesticide toxicity. Yet, it is unknown whether the pesticide impact under DTV is partly ameliorated by a faster pesticide degradation caused by cyclically higher temperatures under DTV. As synergisms may be more likely under energy-limiting conditions, the impact of the pesticide chlorpyrifos was tested under DTV on the mosquito Culex pipiens in the absence and presence of interspecific competition with the water flea Daphnia magna. Chlorpyrifos exposure at a constant temperature without interspecific competition caused considerable mortality, decreased development time, and increased pupal mass of C. pipiens. Competition with D. magna had negative sublethal effects, but it did not affect the toxicity of chlorpyrifos. In contrast, the presence of C. pipiens decreased the impact of chlorpyrifos on D. magna probably due to corporal absorption of chlorpyrifos by C. pipiens. A key finding was that chlorpyrifos no longer caused lethal effects on C. pipiens under DTV, despite DTV on its own being mildly lethal. Additionally, chlorpyrifos exposure under DTV decreased development time less and had no effect anymore on pupal mass compared to chlorpyrifos exposure at a constant temperature. Similarly, the negative chlorpyrifos impact on adult survival of D. magna was less under DTV than at the constant temperature. This could be explained by a faster chlorpyrifos degradation under DTV. This antagonism between pesticide exposure and DTV is likely widespread because organisms experience DTV, many pesticides are applied in pulses, and pesticide degradation is faster at higher temperatures.

Effects of pesticide exposure and predation risk on nutrient cycling and primary production.

Understanding how pesticides and natural stressors shape ecosystem functions remains a major challenge. A largely overlooked way how stressors may affect nutrient cycling and primary production is through effects on body stoichiometry and the egestion of elements. We investigated how exposure to the pesticide chlorpyrifos and to predation risk, an abundant natural stressor in aquatic systems, altered the stoichiometry of the bodies and the egested faecal pellets of Enallagma cyathigerum damselfly larvae and how this further cascaded into effects on primary production (algae growth). Chlorpyrifos exposure reduced egestion rates while predation risk had no effect. Chlorpyrifos exposure and predation risk affected both elemental composition of bodies and faecal pellets, and this in an additive way. Chlorpyrifos exposure increased body C(carbon), N(nitrogen), and P(phosphorous) contents, and increased the C content of the faecal pellets. Predation risk induced an increase of the N content, resulting in a decreased C:N ratio, of both the bodies and faecal pellets. The changes in the composition of the faecal pellets caused by predation risk but not by chlorpyrifos exposure increased algae growth under control conditions. This indicated that algae growth was N limited. Our results provide an important proof-of-principle how a stressor may shape nutrient cycling and subsequently primary productivity.

The effect of warming on pesticide toxicity is reversed between developmental stages in the mosquito Culex pipiens.

A better understanding of interactions between pesticides and warming is important to improve ecological risk assessment in a warming world. Current insights are almost exclusively based on studies that exposed animals simultaneously to both warming and a pesticide and focused on effects during the pesticide exposure period and within a single developmental stage. We studied two ignored aspects of the interplay between warming and pesticide exposure: (i) the role of delayed effects after the pesticide exposure period, and (ii) the dependence on the developmental stage. We carried out a longitudinal experiment from the egg stage to the adult stage in the mosquito Culex pipiens where we crossed a warming treatment (20 °C vs 24 °C) with 48 h exposures to the pesticide chlorpyrifos in three developmental stages (early L1 larvae, late L4 larvae and adults). Chlorpyrifos induced mild to moderate mortality in all developmental stages (10-30%). A key finding was that warming shaped the chlorpyrifos-induced mortality but in opposite directions between stages. Chlorpyrifos was 7% less toxic under warming in L1 larvae, yet more toxic under warming in L4 larvae (22%) and in adult males (33%), while toxicity did not change under warming in adult females. We hypothesize that the general, stage-specific differences in the effects of warming on body size (increased size in early larvae, decreased size in later stages) caused the reversal of the effects of warming on toxicity between stages. Previous larval exposure to chlorpyrifos caused delayed effects that strongly reduced survival to the adult stage (Ì°25% at 24 °C). Notably, warming also modulated these delayed mortality effects in opposite ways between developmental stages, matching the patterns of mortality during the pesticide exposure periods. Integrating the general stage-specific patterns of how warming shapes body size is important to advance our mechanistic understanding of the interactions between pesticides and warming.

Effects of predator cues and pesticide resistance on the toxicity of a (bio)pesticide mixture.

BACKGROUND: Populations of target species are typically exposed to pesticide mixtures and natural stressors such as predator cues, and are increasingly developing resistance to single pesticides. Nevertheless, we have poor knowledge whether natural stressors and the presence of pesticide resistance shape mixture toxicity. We tested the single and combined effects of the pesticide chlorpyrifos and the biopesticide Bacillus thuringiensis israelensis (Bti) on the survival of the Southern house mosquito (Culex quinquefasciatus, Say) and whether these effects were magnified by synthetic predator cues of Notonecta water bugs and differed between a chlorpyrifos-resistant (Ace-1R) and non-resistant (S-Lab) strain. RESULTS: Single exposure to Bti caused mortality in both strains (S-Lab ∼27%, Ace-1R ∼41%) and single exposure to chlorpyrifos caused only mortality in the S-Lab strain (∼33%), while predator cues did not induce mortality. The chlorpyrifos-resistant strain was 1.5-fold more sensitive to Bti, indicating a cost of resistance. The interaction types between chlorpyrifos and Bti (additive), between chlorpyrifos and predator cues (additive), and between Bti and predator cues (synergistic) were consistent in both strains. Despite predator cues making Bti approximately 8% more lethal, they did not change the additive interaction between Bti and chlorpyrifos in their mixture in either strain. CONCLUSION: These results indicate that the resistance against chlorpyrifos was not partly lifted when chlorpyrifos exposure was combined with Bti and predator cues. Identifying the interaction type within pesticide mixtures and how this depends on natural stressors is important to select control strategies that give a disadvantage to resistant individuals compared to non-resistant individuals. © 2019 Society of Chemical Industry.

Resistance to a chemical pesticide increases vulnerability to a biopesticide: Effects on direct mortality and mortality by predation.

Pesticide mixtures are increasingly used to fight pest species that developed resistance to pesticides. To assess the pesticide control efficiency and to reduce ecological damage to non-target species, it is important to quantify the effect of these mixtures and compare them with the effect of their single pesticides on pest species, non-target species and their predator-prey interactions. We studied the effects of the chemical pesticide chlorpyrifos (CPF), the biopesticide Bacillus thuringiensis israelensis (Bti) and their mixture both on the direct mortality and on the mortality by predation. We focused on larvae of a CPF-resistant and a non-resistant strain of the vector mosquito Culex quinquefasciatus and its predator, the pygmy backswimmer Plea minutissima. In the CPF-Bti mixture, both pesticides interacted antagonistically for direct mortality. Exposure to the mixture caused equal direct mortality and equal mortality by predation in both strains. As expected, exposure to CPF resulted in less direct mortality and less mortality by predation in the CPF-resistant mosquito strain compared to the non-resistant strain. Notably, Bti caused a higher mortality in the mosquito larvae of the CPF-resistant strain compared to the non-resistant strain. Furthermore, the predator killed more mosquito larvae of the resistant strain compared to the non-resistant strain when exposed before to Bti alone. These observations identify a novel cost of resistance to a chemical pesticide in terms of increased vulnerability to a biopesticide.

Temperature variation magnifies chlorpyrifos toxicity differently between larval and adult mosquitoes.

To improve risk assessment there is increasing attention for the effect of climate change on the sensitivity to contaminants and vice versa. Two important and connected topics have been largely ignored in this context: (i) the increase of daily temperature variation (DTV) as a key component of climate change, and (ii) differences in sensitivity to climate change and contaminants between developmental stages. We therefore investigated whether DTV magnified the negative effects of the organophosphate insecticide chlorpyrifos on mortality and heat tolerance and whether this effect was stronger in aquatic larvae than in terrestrial adults of the mosquito Culex pipiens. Exposure to chlorpyrifos at a constant temperature imposed mortality and reduced the heat tolerance in both larvae and adult males, but not in adult females. This provides the first evidence that the TICS ("toxicant-induced climate change sensitivity") concept can be sex-specific. DTV had no direct negative effects. Yet, consistent with the CITS ("climate-induced toxicant sensitivity") concept, DTV magnified the toxicity of the pesticide in terms of larval mortality. This was not the case in the adult stage indicating the CITS concept to be dependent on the developmental stage. Notably, chlorpyrifos reduced the heat tolerance of adult females only in the presence of DTV, thereby providing support for the reciprocal effects between DTV and contaminants, hence the coupling of the TICS and CITS concepts. Taken together, our results highlight the importance of integrating DTV and the developmental stage to improve risk assessment of contaminants under climate change.

Additive bioenergetic responses to a pesticide and predation risk in an aquatic insect.

Ignoring natural stressors such as predation risk may contribute to the failure of ecological risk assessment of pesticides to protect freshwater biodiversity. To better understand combined effects of multiple stressors, bioenergetic responses are important as these inform about the balance between energy input and consumption, and provide a unifying mechanism to integrate the impact of multiple stressors with different modes of action. We studied in Enallagma cyathigerum damselfly larvae the single and combined effects of exposure to the pesticide chlorpyrifos and predation risk on life history (survival and growth rate) and bioenergetic response variables at the organismal level (assimilation and conversion efficiency) and the cellular level (cellular energy allocation CEA, energy storage Ea, and energy consumption Ec). Chlorpyrifos exposure almost halved the survival of the damselfly larvae, while predation risk had no effect on survival. Both exposure to the pesticide and to predation risk reduced larval growth rates. This was caused by a reduced conversion efficiency under chlorpyrifos exposure, and by a reduced assimilation efficiency under predation risk. Both chlorpyrifos and predation risk reduced the CEA because of a decreased Ea, and for chlorpyrifos also an increased Ec. The lower Ea was driven by reductions in the fat and glycogen contents. Effects of the pesticide and predation risk were consistently additive and for most variables the strongest response was detected when both stressors were present. The absence of any synergisms may be explained by the high mortality and hypometabolism caused by the pesticide. Our results indicate that CEA can be a sensitive biomarker to evaluate effects of not only contaminants but also natural stressors, such as predation risk, and their combined impact on organisms.

Whether warming magnifies the toxicity of a pesticide is strongly dependent on the concentration and the null model.

How global warming changes the toxicity of contaminants is a research priority at the intersection of global change biology and ecotoxicology. While many pesticides are more toxic at higher temperatures this is not always detected. We studied whether deviations from this general pattern can be explained by concentration-dependent interaction effects and by testing the interaction against the inappropriate null model. We exposed larvae of the mosquito Culex pipiens to three concentrations of the pesticide chlorpyrifos (absence, low and high) in the absence and presence of 4 °C warming. Both the low and high chlorpyrifos concentration were lethal and generated negative sublethal effects: activity of acetylcholinesterase (AChE) and total fat content decreased, and oxidative damage to lipids increased, yet growth rate increased. Warming was slightly lethal, yet had positive sublethal effects: growth rate, total fat content and metabolic rate increased, and oxidative damage decreased. For four out of seven response variables the independent action model identified the expected synergistic interaction between chlorpyrifos and warming. Notably, for three variables (survival, AChE and fat content) this was strongly dependent on the chlorpyrifos concentration, and for two of these (AChE and fat content) not associated with a significant interaction in the general(ized) linear models. For survival and fat content, warming only potentiated chlorpyrifos (CPF) toxicity at the low CPF concentration, while the opposite was true for AChE. Our results highlight that taking into account concentration-dependence and appropriate null model testing is crucial to improve our understanding of the toxicity of contaminants in a warming world.

An adaptive transgenerational effect of warming but not of pesticide exposure determines how a pesticide and warming interact for antipredator behaviour.

The impact of pesticides on organisms may strongly depend on temperature. While many species will be exposed to pesticides and warming both in the parental and offspring generations, transgenerational effects of pesticides under warming are still poorly studied, particularly for behaviour. We therefore studied the single and combined effects of exposure to the pesticide chlorpyrifos (CPF) and warming both within and across generations on antipredator behaviour of larvae of the vector mosquito Culex pipiens. Within each generation pesticide exposure and warming reduced the escape diving time, making the larvae more susceptible to predation. Pesticide exposure of the parents did not affect offspring antipredator behaviour. Yet, parental exposure to warming determined how warming and the pesticide interacted in the offspring generation. When parents were reared at 24 °C, warming no longer reduced offspring diving times in the solvent control, suggesting an adaptive transgenerational effect to prepare the offspring to better deal with a higher predation risk under warming. Related to this, the CPF-induced reduction in diving time was stronger at 20 °C than at 24 °C, except in the offspring whose parents had been exposed to 24 °C. This dependency of the widespread interaction between warming and pesticide exposure on an adaptive transgenerational effect of warming is an important finding at the interface of global change ecology and ecotoxicology.

Daily temperature variation magnifies the toxicity of a mixture consisting of a chemical pesticide and a biopesticide in a vector mosquito.

While many studies on the toxicity of pesticides looked at the effects of a higher mean temperature, effects of the realistic scenario of daily temperature variation are understudied. Moreover, despite the increasing interest for the toxicity of pesticide mixtures how this is influenced by temperature has been largely ignored. We tested whether daily temperature variation (DTV) magnifies the toxicity of two pesticides with a different mode of action, the organophosphate pesticide chlorpyrifos (CPF) and the biopesticide Bacillus thuringiensis var. israelensis (Bti), and of their mixture in the vector mosquito Culex pipiens. Single exposure to CPF and Bti increased mortality and reduced female development time, and exposure to CPF also increased female wing length. DTV was not lethal and did not change the toxicity of the individual pesticides. Yet, a key novel finding was that high DTV increased the mortality of the mixture by changing the interaction between both pesticides from additive to synergistic. Given that in nature daily temperature variation is omnipresent, this is important both for vector control and for ecological risk assessment. The higher toxicity of the mixture at high DTV compared to the typically used constant test temperatures in the laboratory urges caution when evaluating the environmental impact of pesticide mixtures.

Transgenerational interactions between pesticide exposure and warming in a vector mosquito.

While transgenerational plasticity may buffer ectotherms to warming and pesticides separately, it remains unknown how combined exposure to warming and pesticides in the parental generation shapes the vulnerability to these stressors in the offspring. We studied the transgenerational effects of single and combined exposure to warming (4°C increase) and the pesticide chlorpyrifos on life-history traits of the vector mosquito Culex pipiens. Parental exposure to a single stressor, either warming or the pesticide, had negative effects on the offspring: parental exposure to both warming and the pesticide resulted in an overall lower offspring survival, and a delayed offspring metamorphosis. Parental exposure to a single stressor did, however, not alter the vulnerability of the offspring to the same stressor in terms of survival. Parental pesticide exposure resulted in larger offspring when the offspring experienced the same stressor as the parents. Within both the parental and offspring generations, warming made the pesticide more toxic in terms of survival. Yet, this synergism disappeared in the offspring of parents exposed to both stressors simultaneously because in this condition, the pesticide was already more lethal at the lower temperature. Our results indicate that transgenerational effects will not increase the ability of this vector species to deal with pesticides in a warming world. Bifactorial transgenerational experiments are crucial to understand the combined impact of warming and pesticides across generations, hence to assess the efficacy of vector control in a warming world.

A widespread morphological antipredator mechanism reduces the sensitivity to pesticides and increases the susceptibility to warming.

Pollution and predation are two omnipresent stressors in aquatic systems that can interact in multiple ways, thereby challenging accurate assessment of the effects of pollutants in natural systems. Despite the widespread occurrence of morphological antipredator mechanisms, no studies have tested how these can affect the sensitivity of prey to pesticides. Sensitivity to pesticides is typically measured via reductions in growth rates and survival, but also reductions in heat tolerance are to be expected and are becoming increasingly important in a warming world. We investigated how autotomy, a widespread morphological antipredator mechanism where animals sacrifice a body part (here the caudal lamellae) to escape when attacked by a predator, modified the sensitivity to the insecticide chlorpyrifos in larvae of the damselfly Coenagrion puella. Exposure to chlorpyrifos reduced the growth rate and heat tolerance (measured as CTmax). A key finding was that the pesticide had a greater impact on growth rates of intact animals, i.e. those that retained their lamellae. This reduced sensitivity to chlorpyrifos in animals without lamellae can be explained by the reduced outer surface area which is expected to result in a lower uptake of the pesticide. Larvae that underwent autotomy exhibited a lower heat tolerance, which may also be explained by the reduced surface area and the associated reduction in oxygen uptake. There is a wide diversity of morphological antipredator mechanisms, suggesting that there will be more examples where these mechanisms affect the vulnerability to pollutants. Given the importance of pollution and predation as structuring forces in aquatic food webs, exploring the potential interactions between morphological antipredator mechanisms and sensitivity to pollutants will be crucial for risk assessment of pollutants in aquatic systems.

Stronger effects of Roundup than its active ingredient glyphosate in damselfly larvae.

Pesticides are causing strong decreases in aquatic biodiversity at concentrations assumed safe by legislation. One reason for the failing risk assessment may be strong differences in the toxicity of the active ingredient of pesticides and their commercial formulations. Sublethal effects, especially those on behaviour, have been largely ignored in this context, yet can be equally important as lethal effects at the population and ecosystem levels. Here, we compared the toxicity of the herbicide Roundup and its active ingredient glyphosate on survival, but also on ecologically relevant sublethal traits (life history, behaviour and physiology) in damselfly larvae. Roundup was more toxic than glyphosate with negative effects on survival, behaviour and most of the physiological traits being present at lower concentrations (food intake, escape swimming speed) or even only present (survival, sugar and total energy content and muscle mass) following Roundup exposure. This confirms the toxicity of the surfactant POEA. Notably, also glyphosate was not harmless: a realistic concentration of 2mg/l resulted in reduced growth rate, escape swimming speed and fat content. Our results therefore indicate that the toxicity of Roundup cannot be fully attributed to its surfactant, thereby suggesting that also the new generation of glyphosate-based herbicides with other mixtures of surfactants likely will have adverse effects on non-target aquatic organisms. Ecotoxicological studies comparing the toxicity of active ingredients and their commercial formulations typically ignore behaviour while the here observed differential effects on behaviour likely will negatively impact damselfly populations. Our data highlight that risk assessment of pesticides ignoring sublethal effects may contribute to the negative effects of pesticides on aquatic biodiversity.